L-carnitine is an essential substance for mediating long-chain fatty acid transport through the mitochondrian membrane prior to intracellular .beta.-oxidation, and hence a deficiency of L-carnitine causes disorders in fatty acid and its related metabolisms. particularly, it is believed that disorders of the skeletal muscle and cardiac muscle, both of which are high energy consumption tissues depending on carnitine and lacking in carnitine generation, occur from such deficiency. Heretofore a disease arising from inborn irregularities of carnitine metabolism has been studied, however in recent time, secondary disorders of carnitine metabolism have become a problem in patients suffering from nephrosis and undergoing dialysis. Carnitine is administered to carnitine-deficient patients who have a disease of the body muscle or cardiac muscle, or to patients undergoing dialysis. Studies on the behavior of carnitine in disease and therapy have been required, however a desirable assay method for carnitine in the clinical field has not been developed.
Known assay methods for carnitine are as follows:
1. L-carnitine and acetyl CoA are treated with carnitine acetyltransferase (CAT), and the thus-liberated CoASH and 5,5'-dithio-bis-2-nitrobenzoate (DTNB) are further reacted to generate thiophenolate ion which is colorimetrically measured (DTNB method). This method is described in J. Biol. Chem., Vol. 238, p. 2509 (1963), J. Lipid Res., Vol. 5, pp. 184-187 (1964) and Clinical Pathology, Vol. 36, N. 11, pp. 1296-1302 (1988). PA1 2. L-carnitine and .sup.14 C- or .sup.3 H-labelled acetyl CoA are treated with CAT to generate labelled acetyl-L-carnitine and CoASH, and radioactivity is measured (radioisotope method). This method is described in Clin. Chem. Acta, Vol. 37, pp. 235-243 (1972), J. Lipid Res., Vol. 17, pp. 277-182 (1976), and J. Japan. Nut. Food. Soc., Vol. 41, N. 5, pp. 389-395 (1988). PA1 3. L-carnitine and NAD are treated with L-carnitine dehydrogenase to generate 3-dehydrocarnitine and NADH, and increased UV absorption of NADH is measured (carnitine dehydrogenase method). This method is described in Eur. J. Biochem., Vol. 6, pp. 196-201 (1968), ibid. Vol. 10, pp. 56-60 (1969), and Fresenius Z. Anal. Chem., Vol. 320, N. 3, pp. 285-289 (1985). PA1 4. L-carnitine and acetyl CoA are treated with CAT to generate CoA which is reacted with n-{p-(2-benzimidazolyl)-phenyl)-malimide (BIPM), and fluorescent intensity of the resulting CoA-BIPM is measured (fluorescence method). This method is described in Ann. Rep. MHW Institute for Nerve Disease, pp. 315-318 (1986). PA1 (1) L-carnitine dehydrogenase having coenzymes of thionicotinamide adenine dinucleotide group (hereinafter designated thio-NAD group) and nicotinamide adenine dinucleotide group (hereinafter designated NAD group) and which catalyzes a reversible reaction forming essentially dehydrocarnitine from a substrate of L-carnitine, PA1 (2) A.sub.1 and PA1 (3) B.sub.1 PA1 (1) L-carnitine dehydrogenase which has coenzymes of thionicotinamide adenine dinucleotide group (hereinafter designated thio-NAD group) and nicotinamide adenine dinucleotide group (hereinafter designated NAD group) and which catalyzes a reversible reaction forming essentially dehydrocarnitine from a substrate of L-carnitine, PA1 (2) A.sub.1 and PA1 (3) B.sub.1 PA1 wherein A is thio-NAD group or NAD group, when A.sub.1 is thio-NAD group, B.sub.1 is reduced form of NAD group, and when A.sub.1 is NAD group, B.sub.1 is reduced form of thio-NAD group.
In the prior art, the DTNB method and the fluorescence method require a deproteinization treatment in an assay of serum L-carnitine, which is a complex operation. The radioisotope method has an advantage in its sensitivity and specificity, however special facilities are required for measuring radioactivity. The carnitine dehydrogenase method has a disadvantage due to the small molecular absorption coefficient of NADH, i.e. .epsilon.=6.22 (cm.sup.2 /.mu.mol) at 340 nm, and hence it is difficult to assay serum carnitine in a patient disease involving carnitine deficiency (Neurology, 25:16-24 (1975)), and, moreover, the generated NADH is partially consumed by another dehydrogenase pre-existing in serum, such as lactate dehydrogenase, which causes an error in measurement.
We previously invented an assay method of L-carnitine, in which formazan generated in an enzymatic reaction with L-carnitine dehydrogenase was quantitatively measured (Jap. Pat. Appln. No. 1-196550). This method has, however, the disadvantage of insufficient sensitivity for a small amount of serum collected from a premature infant, such as an amount of 20 .mu.l, as compared with a relatively large amount of serum from an adult.
Under these circumstances, it has been desired to develop an advantageous method for assaying L-carnitine without any need for complex treatment such as deproteinization or for special facilities, and which is able to measure even a trace amount of serum L-carnitine taken from a premature infant.